Two mouse homologues of Achaete-Scute Complex genes, Mash-1 and Mash-2, have been isolated using the conservation of the basic-helix-loop-helix domain in this family. Mouse Mash-1 mRNA expression is restricted to cells of the developing central and peripheral nervous systems. There are three successive phases in the distribution of Mash-1 transcripts in the developing central nervous system. Initially, between embryonic day 8.5 and 10.5, Mash-1 transcripts are found in restricted domains in the neuroepithelium of the midbrain and ventral forebrain, as well as in the spinal cord. Between embryonic day 10.5 and 12.5, Mash-1 expression pattern changes from a restricted to a widespread one. Mash-1 transcripts are then found at variable levels in the ventricular zone in all regions of the brain. From embryonic day 12.5 to post-natal stages, Mash-1 is also expressed in cells outside of the ventricular zone throughout the brain. In addition, Mash-1 is expressed during development of the olfactory epithelium and neural retina. Overall, its expression pattern suggest that Mash-1 plays a role at early stages of development of specific neural lineages in most regions of the central nervous system and of several lineages in the peripheral nervous system. Mash-1 and mouse Notch show very similar expression patterns, both spatially and temporally, in the early developing brain and in the retina, suggesting that both genes may participate in the development of the same neural lineages. mash1 is not expressed during the determination of neural precursors and is not required for their specification. Instead, mash1 is present in terminally dividing neural progenitors to control in subsequent differentiation. (Guillemot, 1993).

The expression of murine Mash1 and neurogenin1 (ngn1) have been compared. In the PNS these genes are expressed in complementary autonomic and sensory lineages. In the CNS in situ hybridization to serial sections and double-labeling experiments indicate that Mash1 and ngn1 are expressed in adjacent and nonoverlapping regions of the neuroepithelium that correspond to future functionally distinct areas of the brain. In the PNS several other bHLH genes exhibit similar lineal restriction, as do ngn1 and Mash1, suggesting that complementary cascades of bHLH factors are involved in PNS development. There is a close association between expression of ngn1 and Mash1 and that of two Notch ligands. These observations suggest a basic plan for vertebrate neurogenesis whereby regionalization of the neuroepithelium is followed by activation of a relatively small number of bHLH genes, which are used repeatedly in complementary domains to promote neural determination and differentiation (Ma, 1997).

Mash1 is a transcription factor required during embryogenesis for the development of multiple neural lineages. It is expressed in restricted domains at specific stages in the developing central and peripheral nervous systems and in the developing olfactory epithelium. The regulation of Mash1 has been investigated using transgenic mice containing Mash1/lacZ reporter constructs. Cis acting regulatory elements controlling Mash1 expression in the central nervous system are located within an 8-kb sequence of the Mash1 coding region. This 8-kb sequence does not contain elements directing expression to the peripheral nervous system, olfactory epithelium, or retina. Sequences outside this 8 kb region, but within 36 kb of the Mash1 locus, contain elements responsible for expression in the autonomic division of the peripheral nervous system. However, transgene expression in embryos containing the 36-kb sequence is never detected in the olfactory epithelium and the retina. Thus, regulatory elements driving expression in these lineages may be at even greater distances from the Mash1 coding region (Verma-Kurvari, 1997).

Mash1, a transcription factor of the basic helix-loop-helix class, is expressed during embryogenesis in restricted regions of the nervous system. An essential role for Mash1 in neural development has been previously demonstrated in mice carrying a targeted disruption of the Mash1 gene. Regulation of the precise temporal and spatial expression of Mash1 is thus likely to be important for proper neural development. Sequences that regulate Mash1 expression in the central nervous system have been characterized by assaying the expression of lacZ reporter genes in transgenic embryos. A 1158-bp enhancer localized approximately 7 kb upstream of the Mash1 coding region has been identified. Deletions within this enhancer region reveal the presence of both positive and negative cis-acting elements. Analysis of multiple sequences within the enhancer demonstrate that different elements preferentially function in different regions within the Mash1-specific CNS expression domain. Specific sequences confering expression in the hypothalamic region of the diencephalon and the dorsal mesencephalon have been identified, and a region having an essential role for the whole enhancer region has been localized. A role for sequences 3' downstream of the Mash1 coding region has been identified, providing evidence for posttranscriptional control of Mash1 expression in multiple CNS domains (Verma-Kurvari, 1998).

Mash1, a neural-specific bHLH transcription factor, is essential for the formation of multiple CNS and PNS neural lineages. Transcription from the Mash1 locus is elevated in mice null for Mash1, suggesting that MASH1 normally acts to repress its own transcription. This activity is contrary to the positive autoregulation of other proneural bHLH proteins. To investigate the mechanisms involved in this process, sequences flanking the Mash1 gene were tested for the ability to mediate negative autoregulation. A Mash1/lacZ transgene containing 36 kb of cis-regulatory sequence exhibits an increase in lacZ expression in the Mash1 mutant background, which phenocopies the observation of transcriptional autoregulation at the endogenous Mash1 locus. Using Mash1/lacZ lines with progressively less cis-acting sequence, autoregulatory responsive elements have been demonstrated to colocalize with a previously characterized 1.2-kb CNS enhancer. Mutations of E-box sites within this enhancer do not result in an apparent loss of autoregulation, suggesting that MASH1 does not directly repress its own transcription. Interestingly, these mutations do not indicate any underlying positive auto- or cross-regulation of Mash1. Furthermore, the loss of autoregulation in the Mash1 mutant background is reminiscent of a loss of lateral inhibitory signaling. However, mutations in Hairy/Enhancer-of-split-like (HES) consensus sites, the likely purveyors of Notch-mediated lateral inhibition, do not support a role for these sites in negative autoregulation. It is hypothesized that MASH1 normally inhibits its own expression indirectly, possibly through a HES-mediated repression of positive regulators or through novel HES binding sites (Meredith, 2000).

Within the developing vertebrate nervous system, specific subclasses of neurons are produced in vastly different numbers at defined times and locations. This implies the concomitant activation of a program that controls pan-neuronal differentiation and of a program that specifies neuronal subtype identity, but how these programs are coordinated in time and space is not well understood. Loss- and gain-of-function studies have defined Phox2b as a homeodomain transcription factor that coordinately regulates generic and type-specific neuronal properties. It is necessary and sufficient to impose differentiation towards a branchio- and viscero-motoneuronal phenotype and at the same time promote generic neuronal differentiation. The underlying genetic interactions have been examined. Phox2b has a dual action on pan-neuronal differentiation. It upregulates the expression of proneural genes (Ngn2) when expressed alone and upregulates the expression of Mash1 when expressed in combination with Nkx2.2. By a separate pathway, Phox2b represses expression of the inhibitors of neurogenesis Hes5 and Id2. The role of Phox2b in the specification of neuronal subtype identity appears to depend in part on its capacity to act as a patterning gene in the progenitor domain. Phox2b misexpression represses the Pax6 and Olig2 genes, which should inhibit a branchiomotor fate, and induces Nkx6.1 and Nkx6.2, which are expressed in branchiomotor progenitors. Phox2b behaves like a transcriptional activator in the promotion of both, generic neuronal differentiation and expression of the motoneuronal marker Islet1. These results provide insights into the mechanisms by which a homeodomain transcription factor through interaction with other factors controls both generic and type-specific features of neuronal differentiation (Dubreuil, 2002).

Patterning of the dorsal neural tube involves Bmp signaling, which results in activation of multiple pathways leading to the formation of neural crest, roof plate and dorsal interneuron cell types. Constitutive activation of Bmp signaling at early stages (HH10-12) of chick neural tube development induces roof-plate cell fate, accompanied by an increase of programmed cell death and a repression of neuronal differentiation. These activities are mimicked by the overexpression of the homeodomain transcription factor Msx1, a factor known to be induced by Bmp signaling. By contrast, the closely related factor, Msx3, does not have these activities. At later stages of neural tube development (HH14-16), dorsal progenitor cells lose their competence to generate roof-plate cells in response to Bmp signaling and instead generate dorsal interneurons. This aspect of Bmp signaling is phenocopied by the overexpression of Msx3 but not Msx1. Taken together, these results suggest that these two different Msx family members can mediate distinct aspects of Bmp signaling during neural tube development (Liu, 2004).

Two lines of evidence suggest that the neural bHLH genes that are crucial for neuronal differentiation might be direct transcriptional targets of Msx1. (1) In pursuit of factors that regulate the expression of the neural bHLH genes by yeast one-hybrid screening, Msx1 was identified to be potential regulator for both Math1 and Mash1 and several consensus sites for Msx1 binding are present in the enhancer regions of Math1/Cath1 and Mash1/Cash1. (2) Both Msx1 and Msx3 can bind to these consensus sites in vitro. However, because in vivo Msx1 represses the bHLH factor expression and Msx3 induces Cath1 expression, additional in vivo co-factors or chromatin properties that modulate these activities must be invoked (Liu, 2004).

Induction of the sympathetic nervous system (SNS) from its neural crest (NC) precursors is dependent on BMP signaling from the dorsal aorta. To determine the roles of BMP signaling and the pathways involved in SNS development, components of the BMP pathways were conditionally knocked out. To determine if BMP signaling is a cell-autonomous requirement of SNS development, the Alk3 (BMP receptor IA) was deleted in the NC lineage. The loss of Alk3 does not prevent NC cell migration, but the cells die immediately after reaching the dorsal aorta. The paired homeodomain factor Phox2b, known to be essential for survival of SNS precursors, is downregulated, suggesting that Phox2b is a target of BMP signaling. To determine if Alk3 signals through the canonical BMP pathway, Smad4 was deleted in the NC lineage. Loss of Smad4 does not affect neurogenesis and ganglia formation; however, proliferation and noradrenergic differentiation are reduced. Analysis of transcription factors regulating SNS development shows that the basic helix-loop-helix factor Ascl1 is downregulated by loss of Smad4 and that Ascl1 regulates SNS proliferation but not noradrenergic differentiation. To determine if the BMP-activated Tak1 (Map3k7) pathway plays a role in SNS development, Tak1 was deleted in the NC lineage. Tak1 was shown not to be involved in SNS development. Taken together, these results suggest multiple roles for BMP signaling during SNS development. The Smad4-independent pathway acts through the activation of Phox2b to regulate survival of SNS precursors, whereas the Smad4-dependent pathway controls noradrenergic differentiation and regulates proliferation by maintaining Ascl1 expression (Morikawa, 2009).

Neural precursors in the developing olfactory epithelium (OE) give rise to three major neuronal classes - olfactory receptor (ORNs), vomeronasal (VRNs) and gonadotropin releasing hormone (GnRH) neurons. Nevertheless, the molecular and proliferative identities of these precursors are largely unknown. Two precursor classes were characterized in the olfactory epithelium (OE) shortly after it becomes a distinct tissue at midgestation in the mouse: slowly dividing self-renewing precursors that express Meis1/2 at high levels, and rapidly dividing neurogenic precursors that express high levels of Sox2 and Ascl1. Precursors expressing high levels of Meis genes primarily reside in the lateral OE, whereas precursors expressing high levels of Sox2 and Ascl1 primarily reside in the medial OE. Fgf8 maintains these expression signatures and proliferative identities. Using electroporation in the wild-type embryonic OE in vitro as well as Fgf8, Sox2 and Ascl1 mutant mice in vivo, it was found that Sox2 dose and Meis1 -- independent of Pbx co-factors -- regulate Ascl1 expression and the transition from lateral to medial precursor state. Thus, proliferative characteristics and a dose-dependent transcriptional network were characterized that define distinct OE precursors: medial precursors that are most probably transit amplifying neurogenic progenitors for ORNs, VRNs and GnRH neurons, and lateral precursors that include multi-potent self-renewing OE neural stem cells (Tucker, 2010).

Proneural proteins play a central role in vertebrate neurogenesis, but little is known of the genes that they regulate and of the factors that interact with proneural proteins to activate a neurogenic program. The proneural protein Mash1 and the POU proteins Brn1 and Brn2 interact on the promoter of the Notch ligand Delta1 and synergistically activate Delta1 transcription, a key step in neurogenesis. Overexpression experiments in vivo indicate that Brn2, like Mash1, regulates additional aspects of neurogenesis, including the division of progenitors and the differentiation and migration of neurons. This study identifies, by in silico screening, a number of additional candidate target genes that are recognized by Mash1 and Brn proteins through a DNA-binding motif similar to that found in the Delta1 gene and present a broad range of activities. It is thus proposed that Mash1 synergizes with Brn factors to regulate multiple steps of neurogenesis (Castro, 2006).

Delta1 is a common target of the proneural genes Mash1 and Neurogenin1/2 in mouse embryos. To determine whether Mash1 and Neurogenin1/2 directly transcribe Delta1, the regulatory sequences of this gene were analyzed. Two evolutionarily conserved enhancers active in different CNS regions have been identified in the Delta1 gene. To determine whether these enhancers mediate the regulation of Delta1 by proneural genes, transgenic mouse lines were generated. A transgene containing the full-length 4.3 kb mouse Delta1 promoter driving lacZ was expressed broadly in the embryonic brain and spinal cord at E11.5, in a pattern similar to that of endogenous Delta1. A transgene containing the proximal Delta1 neural enhancer (hereafter called DeltaM) and a minimal promoter driving lacZ was, in contrast, only expressed in parts of the Delta1 expression domain, including the dorsal spinal cord and ventral telencephalon, which also express Mash1. A transgene containing the distal Delta1 enhancer (hereafter called DeltaN) driving lacZ was expressed in a complementary manner in the neural tube, including the ventral spinal cord and dorsal telencephalon, which also express Ngn1 and Ngn2. To test whether these enhancers are regulated by proneural genes, the transgenic lines were bred with proneural null mutant mice. On a Mash1 null mutant background, the DeltaM-lacZ transgene was not expressed in the CNS at E11.5, demonstrating that the DeltaM enhancer requires Mash1 function for its activation. On a Ngn1;Ngn2 double mutant background, the DeltaN-lacZ transgene showed reduced expression in the spinal cord and was not expressed in the brain, except near the hindbrain border, showing that the DeltaN enhancer is activated by Neurogenins (Castro, 2006).

It was next asked whether proneural proteins directly interact with the Delta1 enhancers in the embryonic CNS by performing chromatin immune precipitation (ChIP) experiments. An antibody to Mash1 coprecipitated the DeltaM sequence in chromatin prepared from E12.5 wild-type telencephalon, but not from Mash1 mutant telencephalon, and the antibody did not precipitate the DeltaN sequence or the Delta1 coding sequence. Conversely, an antibody to Ngn2 coprecipitated the DeltaN sequence from wild-type, but not from Ngn2 null mutant telencephalon, and it did not precipitate the DeltaM or Delta1 coding sequences. Therefore, Mash1 and Ngn2 specifically bind in vivo to the DeltaM and DeltaN enhancers, respectively (Castro, 2006).

To further examine the interaction of Mash1 with the DeltaM enhancer, transcription assays were performed in P19 cells. It was first verified that Mash1 induces Delta1 transcription in these cells. After transfection of P19 cells with a Mash1 expression vector, the Mash1 transcript level increased in less than 4 hr, while the Delta1 transcript level increased less than 3 hr later, suggesting that Delta1 is directly transcribed by Mash1 in this system. By performing ChIP experiments with the Mash1 antibody on Mash1-transfected and mock-transfected P19 cells, it was shown that Mash1 specifically binds to the DeltaM sequence, indicating that Mash1 uses the same enhancer element to activate Delta1 in P19 cells and in the embryo. To examine the regulation of DeltaM by Mash1, the DeltaM sequence was inserted in a luciferase reporter vector and its transcriptional activity was tested in P19 cells. The DeltaM reporter was strongly activated when cotransfected with a Mash1 expression construct (Castro, 2006).

Two E-boxes (hereafter called E1-box and E2-box) were identified in the DeltaM sequence that are completely conserved in the human, mouse, chick, and zebrafish Delta1/DeltaD gene. To test whether these motifs mediate the direct binding of Mash1 to DeltaM, the two E-boxes were mutated either separately or together and examined the activity of the resulting DeltaM mutants. Mutation of each E-box separately or the two E-boxes together abolished activation of DeltaM by Mash1 and severely reduced the capacity of Mash1 to activate the full-length Delta1 promoter in P19 cells. A shorter version of DeltaM that mostly contains the 2 E-boxes and the 17 nucleotides in between (DeltaM short was activated by Mash1 as efficiently as DeltaM (Castro, 2006).

To determine whether activation of DeltaM by Mash1 involves motifs other than the two E-boxes, the DeltaM short element was mutated further. Interestingly, a perfect evolutionarily conserved consensus binding site for the POU family of homeodomain proteins, or octamer, is present in this element, one nucleotide 5′ from the E2-box. Point mutations in each half-site of the octamer motif, oct^T64G and oct^C68G, known to disrupt the interaction of the octamer with the homeodomain (POU_H) and the POU-specific domain (POU_S) of POU factors, respectively, abolished activation of DeltaM short by Mash1, and the same mutations introduced in the complete DeltaM element or the full-length Delta1 promoter also severely reduced their activation by Mash1. This raised the possibility that activation of Delta1 by Mash1 requires binding of both Mash1 and a POU protein to adjacent motifs in the DeltaM enhancer (Castro, 2006).

The proximity of the two sites suggested that binding of Mash1 to the E2-box might be influenced by binding of a putative POU protein to the adjacent octamer. To address this possibility, a luciferase reporter vector containing the multimerized E2 sequence and a minimal promoter (E2₆ construct) was generated, so that the interaction of Mash1 with E2 could be analyzed in P19 cells independently of the rest of the DeltaM element. It was asked whether the octamer sequence adjacent to E2 had an effect on the Mash1::E2 interaction by generating a reporter construct containing three copies of a sequence containing both the octamer and the E2-box in the same configuration as in DeltaM ([oct+E2]₃). Mash1 activated this construct more efficiently. Moreover, a mutation in the octamer that interferes with POU protein binding (oct^T64G abolished activation of (oct^mut+E2)₃ by Mash1, thus suggesting that binding of a POU protein to the octamer sequence increases the efficiency of the Mash1::E2 interaction (Castro, 2006).

This study shows that the activation of Delta1 expression by Mash1, a key aspect of its proneural function, involves a functional synergy between Mash1 and the POU genes Brn1 and Brn2. The synergistic activation of Delta1 by Mash1 and Brn1/2 likely reflects recruitment of Mash1 by a Brn protein to the DeltaM enhancer. Brn1/2 proteins on their own bind strongly to the consensus octamer sequence present in this enhancer, while Mash1 alone binds only poorly to the adjacent E2-box, but Mash1 efficiently forms a complex with Brn proteins on the octamer-E2 motif. The configuration of this binding motif plays an essential role in the recruitment process, since increasing the distance between the octamer and the E2-box by just one nucleotide is sufficient to abolish Mash1 recruitment and enhancer activity. The importance of keeping the two DNA-binding sites in close proximity strongly suggests that Mash1-E47 and Brn1/2 physically interact when bound to DNA (Castro, 2006).

The interaction of Mash1 and Brn proteins may also enhance the transcriptional activity of the complex. This is another well-documented mechanism of functional synergy, operating, for example, in the interaction between NeuroM, Isl1, and Lhx3 on the HB9 promoter. Although the primary mechanism underlying the functional synergy of Mash1 and Brn1/2 on the Delta1 promoter is cooperative binding to DNA, the role of Brn proteins does not appear to be restricted to Mash1 recruitment. Indeed, direct binding of Mash1 to the Mash1/Brn motif in the absence of Brn protein binding, e.g., when the low-affinity E2-box sequence in the Mash1/Brn motif is converted to a high-affinity one, is not sufficient to activate a Delta1 reporter construct. This suggests that Brn1/2 also potentiate the transcriptional activity of Mash1, perhaps by recruiting an essential coactivator or by initiating a conformational change that exposes the Mash1 activation domain (Castro, 2006).

An evolutionarily conserved Mash1/Brn-binding motif was found in the vicinity of 21 mouse genes. Six of them are components of the Notch pathway, which, together with the finding that a dominant-negative Brn construct blocks Notch activity in the chick neural tube, suggests that Mash1/Brn protein complexes play a major role in regulating Notch signaling in the CNS. Other genes associated with a Mash1/Brn motif also have important roles in neural development but act independently of Notch signaling. This is notably the case of Dcamkl1 or doublecortin-like kinase, a microtubule-associated protein that has recently been implicated in multiple aspects of development of the cerebral cortex, including cell cycle progression, neuronal commitment, neuronal migration, and axon growth (Castro, 2006).

Some of the other genes associated with a Mash1/Brn motif have not been previously studied in the developing nervous system, but studies in other systems suggest that they may also have varied functions during neurogenesis downstream of Mash1 and Brn1/2. The zinc finger transcription factor Insm1 is regulated by the bHLH gene Neurogenin3 in the pancreas, where it promotes neuroendocrine cell differentiation. Fbw7 is an ubiquitin ligase with an important role in promoting cell cycle arrest in G1/G0 through degradation of cyclin E, c-myc, and c-jun. Fbw7 has also been implicated in degradation of Notch1 (Castro, 2006).

These data thus support the idea that Mash1 acts in synergy with Brn proteins to activate a genetic program that controls multiple steps of neurogenesis, including precursor selection through Notch activation, cell cycle exit, neuronal differentiation, and migration. Analysis of Brn1/Brn2 double mutant mice has shown that these two genes regulate neuronal migration and the proliferation of subventricular zone precursors in the cerebral cortex, a region where neurogenesis is primarily regulated by the proneural gene Ngn2. Whether Brn1/Brn2 mutant mice also display neurogenesis defects in regions where Mash1 is the main proneural gene remains to be analyzed (Castro, 2006).

An important question raised by these results is whether Mash1 regulates aspects of neurogenesis independently of Brn proteins. In support of this notion, additional direct targets of Mash1 have been identified in the brain that are not associated with a conserved Mash1/Brn motif. Moreover, a study of Mash1 function in a neuroendocrine prostate cell line has revealed a number of putative direct targets in this tissue that are not associated with a conserved Mash1/Brn motif, and some of these genes are also regulated by Mash1 in the telencephalon in overexpression experiments. These different findings thus support a model whereby Mash1 interacts with different DNA-binding cofactors to activate different subprograms of neurogenesis, similar to the regulation of different subprograms of myogenesis by MyoD (Castro, 2006).

Cellular differentiation and lineage commitment are considered to be robust and irreversible processes during development. Recent work has shown that mouse and human fibroblasts can be reprogrammed to a pluripotent state with a combination of four transcription factors. This raised the question of whether transcription factors could directly induce other defined somatic cell fates, and not only an undifferentiated state. It was hypothesized that combinatorial expression of neural-lineage-specific transcription factors could directly convert fibroblasts into neurons. Starting from a pool of nineteen candidate genes, a combination of only three factors, Ascl1, Brn2 (also called Pou3f2) and Myt1l, was found to suffice to rapidly and efficiently convert mouse embryonic and postnatal fibroblasts into functional neurons in vitro. These induced neuronal (iN) cells express multiple neuron-specific proteins, generate action potentials and form functional synapses. Generation of iN cells from non-neural lineages could have important implications for studies of neural development, neurological disease modelling and regenerative medicine (Vierbuchen, 2010).

Switching specific patterns of gene repression and activation in response to precise temporal/spatial signals is critical for normal development. This study reports a pathway in which induction of CaMKII triggers an unexpected switch in the function of the HES1 transcription factor from a TLE-dependent repressor to an activator required for neuronal differentiation. These events are based on activation of the poly(ADP-ribose) polymerase1 (PARP-1) sensor component of the groucho/TLE-corepressor complex mediating dismissal of the corepressor complex from HES1-regulated promoters. In parallel, CaMKII mediates a required phosphorylation of HES1 to permit neurogenic gene activation, revealing the ability of a specific signaling pathway to modulate both the derepression and the subsequent coactivator recruitment events required for transcriptional activation of a neurogenic program. The identification of PARP-1 as a regulated promoter-specific exchange factor required for activation of specific neurogenic gene programs is likely to be prototypic of similar molecular mechanisms (Ju, 2004).

A covalent modification recently linked to transcription is poly(ADP-ribosyl)ation of proteins mediated by the poly(ADP-ribose) polymerase1 (PARP-1) enzyme. PARP-1 catalyzes the transfer of ADP-ribose chains onto glutamic acid residues of acceptor proteins, including itself (automodification), histones, transcription factors, and DNA repair proteins using NAD+ as a substrate involved in chromatin decondensation, DNA replication, and DNA repair. Therefore, poly(ADP-ribosyl)ation by PARP-1 affects cellular processes such as apoptosis, necrosis, cellular differentiation, malignant transformation, and modulations activities of transcription factors. While it has been recently reported that PARP influences both the expression and silencing at diverse times during Drosophila development (Tulin, 2002), it has been demonstrated that high PARP enzymatic activity is observed in areas of high transcriptional activity and chromatin decondensation on the polytene chromatin (Tulin, 2003). Together these observations suggest that PARP-1 may exert its function in transcription through direct binding to the gene-regulating sequences and through modification of transcription factors by poly(ADP-ribosyl)ation (Ju, 2004).

This study finds that HES1-dependent repression of MASH1 is dependent upon the actions of the TLE1 corepressor complex. Not only are additional insights provided into the molecular mechanisms of TLE1-mediated repression but also the molecular mechanism of the switch to activation function has been uncovered. The composition of this TLE1 complex is distinct from those of other reported corepressor complexes such as N-CoR/SMRT and CtBP. Interestingly, roles for transcriptional regulation and chromatin remodeling activities have been described for most of the components of the TLE1 complex identified, but no component alone is indispensable for at least some level of TLE1-mediated repression (Ju, 2004).

Consistent with the observation that the enzymatic activity of PARP-1 is not required for HES1-mediated MASH1 repression, these data favors a model predicting that in the TLE1 holorepressor complex, the enzymatic activity of PARP-1 is inhibited. Exposure to a signal inducing neuronal differentiation causes activation of CaMKIIdelta, which is proven to be required for activation of neurogenic genes. Once sufficient levels of CaMKIIdelta are achieved (5-7 hr), it will, directly or indirectly, mediate phosphorylation and activation of PARP-1, which then catalyzes poly(ADP-ribosyl)ation of TLE1 and most of the other components to the corepressor complex. This is consistent with the observation that calcium signaling evoked by extrinsic and intrinsic cues can induce auto-poly(ADP-ribosyl)ation of PARP-1; however, CaMKIIdelta may also be activated in a calcium-independent fashion. This covalent modification is suggested to result in their dismissal from the biochemical complex and derepression of the MASH1 gene. The role of PARP-1 in derepression of MASH1 and its retention on the activated MASH1 promoter is quite consistent with reports that poly(ADP-ribosyl)ation of chromatin-associated proteins induce major changes in chromosomal architecture. However, in the case of MASH1, it was found that derepression alone is insufficient for induction. This is in accord with the findings for other regulated transcription factors. For example, the loss of the N-CoR corepressor is not alone sufficient to activate most AP-1-regulated genes, and only in a subset of RAR target genes does derepression result in a signal-independent 'default' activation of gene targets.

These data also indicate that, in addition to Ca²⁺-CaMKII-dependent dissociation of the TLE1 corepressor from HES1, a covalent modification of HES1 itself is required to permit activation of MASH1. Thus, activation of MASH1 is linked to sequential CaMKIIdelta-dependent activation of PARP-1 enzymatic activity, which was previously inhibited in the TLE1 holocorepressor complex, permitting dismissal of the TLE1 complex, derepression and phosphorylation of HES1, and recruitment of specific coactivators and thus causing and maintaining derepression of genes mediating neuronal differentiation. The actions of PARP-1 in the TLE1-mediated events are thus analogous to the effects of covalent modifications by phosphorylation and acetylation, as mediators of switches from repressor to activator function (Ju, 2004).

While HES1 is recognized to regulate tissue morphogenesis by maintaining undifferentiated cells and preventing differentiation, continued occupancy of the MASH1 promoter by HES1 in the differentiating neural stem cells has surprisingly proven to be required to initiate MASH1 activation events. Indeed, previous, seemingly contradictory reports of transient transfection assays in which HES1 can inhibit acid beta-glucosidase genes in HepG2 cells but cause activation in fibroblasts are likely to be explained by findings of signal-dependent HES1 switching events (Ju, 2004).

This cortical progenitor culture system has permitted identification of a regulatory pathway that may be, at least in part, partially compensated in vivo because many nestin-positive neural stem cells in the subventricular zone proliferate without losing the multipotentiality to differentiate into neurons in HES1 mutant or even HES1/HES5 double mutant mice. It is suggested that there may be additional HES1-like repressors or unidentified protein partners, including HERP and other E box binding proteins, that are also potentially involved in MASH1 gene activation. The identification of this unexpected mechanism of HES1 action in cortical progenitor cell cultures suggest that, in this system, the other molecules that could assume a similar function were either not expressed or required. The finding of a requirement of HES1 in activation of neurogenic genes is consistent with suggestions that HES1 might promote differentiation, in addition to its role in maintenance of the undifferentiated state, at multiple steps of neural stem cell development (Ju, 2004).

In summary, a pathway is suggested in which a PARP-1-containing TLE1 complex is recruited by the Notch-induced bHLH factor, HES1, initially mediating repression of MASH1 in the proliferating neural stem cells. The data suggest that signals that induce neuronal differentiation, such as PDGF in neural stem cells, act to induce the CaMKIIdelta isoform, which, in turn, is required for HES1-mediated MASH1 activation . The temporal aspects of CaMKIIdelta induction appear to account for the delay in derepression and activation of MASH1 expression following critical PDGF signaling. CaMKIIdelta-induced phosphorylation of a specific serine residue in the orange domain of HES1 permits it to recruit coactivators, including CBP, and has proven to be required for activation of the MASH1. The conserved relationship of the HES1 orange domain with the HLH domain raises the possibility of an additional role in protein interactions that include dimerization (Ju, 2004).

In a sense, the observation that a component of the TLE1-mediated repression complex, PARP-1, is also required for derepression events and maintenance of activation, parallels the requirement of TBL1/TBLR1 complex for ligand-dependent exchange of N-CoR corepressor complexes for coactivators in the switch of nuclear receptor function from repression to activation. TBLR1 is required for recruitment of the ubiquitylation/19S proteosome complex to prevent N-CoR/SMRT-dependent maintenance of a repression checkpoint. It is suggested that PARP-1 may achieve the same effects by a distinct modification strategy and serves as a regulated sensor of neuron-inducing signals based on the actions of CaMKIIdelta induced by the initial stimulus to neuronal differentiation. Therefore, it is tempting to speculate that PARP-1 and TBLR1 may be critical for the exchange events required to overcome the repression checkpoint for TLE- and N-CoR-regulated repressors, respectively (Ju, 2004).

The dual functions of PARP-1 and HES1 in the progression of neural stem cells along a neuronal pathway indicate that while the initial Notch signal causes repression of neurogenic genes by induction of HES1, it simultaneously arms the response to subsequent Ca²⁺-CaMKII signals that permit MASH1 gene activation events. The data illustrate how a single signaling pathway can mediate a sequential, two-step derepression/activation process required for development in gene activation. Induction of a Ca²⁺/CaMKII-dependent program initiates both PARP-1 activation, which is required for dismissal of the TLE1 corepressor complex, and a second event, covalent modification of HES1, which is required for target gene activation. The requirement for both a derepression and independently mediated activation event is likely to be prototypic of many similar functions of PARP-1 factors in development. The sequential calcium-regulated PARP-1-dependent switch from repression to derepression to activation function of HES1 is clearly an effective strategy to maximize the amplitude of the transcriptional response of neurogenic gene expression to signals and to permit temporally precise patterns of cellular response (Ju, 2004).

Establishment of neuronal networks is an extremely complex process involving the interaction of a diversity of neuronal cells. During mammalian development, these highly organized networks are formed through the differentiation of multipotent neuronal progenitors into multiple neuronal cell lineages. In the developing forebrain of mammals, the combined function of the Dlx1, Dlx2, Dlx5 and Dlx6 homeobox genes is necessary for the differentiation of the GABAergic interneurons born in the ventricular and subventricular zones of the ventral telencephalon, as well as for the migration of these neurons to the hippocampus, cerebral cortex and olfactory bulbs. The 437 bp I12b enhancer sequence in the intergenic region of the Dlx1/2 bigene cluster is involved in the forebrain regulation of Dlx1/2. Using DNase I footprinting, six regions of I12b potentially bound by transcription factors were identified. Mutagenesis of each binding site affected the expression of reporter constructs in transgenic mice. However, the effects of impairing protein-DNA interactions were not uniform across the forebrain Dlx1/2 expression domains, suggesting that distinct regulatory interactions are taking place in the different populations of neuronal precursors. Analyses of protein-DNA interactions provide evidence of a direct role for MASH1 in Dlx1/2 regulation in the forebrain. DLX proteins play a crucial role in the maintenance of their own expression, as shown by transgenic and co-transfection experiments. These studies suggest that the seemingly continuous domains of Dlx gene expression in the telencephalon and diencephalon are in fact the combination of distinct cell populations within which different genetic regulatory interactions take place (Poitras, 2007).

Proneural genes such as Ascl1 are known to promote cell cycle exit and neuronal differentiation when expressed in neural progenitor cells. The mechanisms by which proneural genes activate neurogenesis--and, in particular, the genes that they regulate--however, are mostly unknown. A genome-wide characterization of the transcriptional targets of Ascl1 in the embryonic brain and in neural stem cell cultures was carried out by location analysis and expression profiling of embryos overexpressing or mutant for Ascl1. Identified targets included Sox4, Dlx2, Ebf3, Gli3, and Nf1b. The wide range of molecular and cellular functions represented among these targets suggests that Ascl1 directly controls the specification of neural progenitors as well as the later steps of neuronal differentiation and neurite outgrowth. Surprisingly, Ascl1 also regulates the expression of a large number of genes involved in cell cycle progression, including canonical cell cycle regulators and oncogenic transcription factors. Mutational analysis in the embryonic brain and manipulation of Ascl1 activity in neural stem cell cultures revealed that Ascl1 is indeed required for normal proliferation of neural progenitors. This study identified a novel and unexpected activity of the proneural gene Ascl1, and revealed a direct molecular link between the phase of expansion of neural progenitors and the subsequent phases of cell cycle exit and neuronal differentiation (Castro, 2011).

The most unexpected finding from this study is that Ascl1 activates a large number of positive cell cycle regulators in the embryonic telencephalon and NS cells. Ascl1 target genes include several canonical cell cycle regulators that are essential for G1/S transition (e.g., E2f1, Cdk1, Cdk2, and Skp2) or entry into mitosis (e.g., Cdk1 and Cdc25b). Other Ascl1 targets have additional roles besides cell cycle control. FoxM1 has an important role in progression of normal and tumor cells through M phase and also promotes angiogenesis through activation of vascular endothelial growth factor (VEGF) expression. Cyr61/Ccn1, a secreted heparin-binding protein, also promotes both cell proliferation and angiogenesis. Ascl1 therefore promotes cell cycle progression at both G1/S and G2/M transitions, and may also coordinate cell cycle regulation with other aspects of the biology of neural progenitors. Ascl1 shares some of its targets with c-Myc and N-Myc—including Birc5, Cdca7, Ccnd2, Foxm1, and the Notch ligand Dll3 - suggesting that its role in mitotic progenitors may involve cooperation with Myc proteins. In the absence of Ascl1, Myc proteins may be able to sustain the division of certain progenitors (e.g., in the telencephalic VZ) (Castro, 2011).

Direct targets of Ascl1 in the embryonic telencephalon also include genes involved in cell cycle arrest—including Fbxw7, Gadd45g, Ccng2, Hipk2, and Prmt2—thus demonstrating that Ascl1 regulates two sets of targets with opposite roles in cell cycle control. Interestingly, several of the cell cycle arrest targets have distinct spatial and/or temporal expression patterns from those of cell cycle progression targets in both the embryonic brain and NS5 cells, suggesting that they are regulated during a later phase of neurogenesis. Two findings support this assertion. First, cell cycle arrest targets are strongly up-regulated when NS5 cells are induced to differentiate by overexpression of Ascl1, and they are not affected by overexpression of DN-Ascl1. Second, their transcripts are found mostly outside of the VZ in the embryonic telencephalon. Gadd45g and Fbxw7 transcripts are localized to the SVZ and may be expressed by progenitor cells that are going through their last division or are already post-mitotic. Ccng2 and Prmt2 transcripts are present mostly in the MZ, where these genes may prevent cell cycle re-entry of post-mitotic neurons (Castro, 2011).

Together, these results suggest that Ascl1 has a role in both cell cycle promotion and cell cycle termination, and that the transition between these two functions involves the coordinated down-regulation and induction of multiple target genes. It is unusual that the same TF promotes in the same lineage both the expansion of progenitors and their subsequent division arrest and differentiation. Interestingly, Asense, the Ascl1 ortholog in Drosophila, has been shown recently to promote neuroblast self-renewal, while earlier studies had suggested a role of Asense in inhibiting cell divisions of neuroblast daughter cells, suggesting that this dual activity is an evolutionarily conserved feature of the Ascl1 gene family. A selective advantage may be that it allows for a rapid and efficient switch from a proliferating progenitor state to a post-mitotic neuronal state and ensures that the regulatory programs that underpin these two states are mutually exclusive (Castro, 2011).

The processes that select which set of target genes Ascl1 regulates in progenitors versus differentiating neurons are not currently known. A differential response of cell cycle progression genes and cell cycle arrest genes to different levels of Ascl1 is ruled out by the observation that Ascl1 protein levels do not increase substantially when NS cells differentiate. Other mechanisms, however, can be envisaged. The differential binding of Ascl1 to the Ccng2 and Foxm1 promoters in proliferating versus differentiating NS5 cells suggests that changes in Ascl1 structure (e.g., post-translational modifications such as phosphorylation) and/or Ascl1 regulation (e.g., a switch from oscillatory to stable expression) control Ascl1-promoter interactions and possibly other aspects of target gene regulation. Other pathways coregulating Ascl1 targets may also contribute to the change in the Ascl1 transcriptional program as neural progenitors stop proliferating and begin to differentiate. The enrichment of a consensus motif for CBF1/RBPj binding in promoters of cell cycle progression targets raises the intriguing possibility that coregulation by Ascl1 and the Notch pathway results in expression of these genes in proliferating progenitors where Notch is signaling and their repression in differentiating neurons where the Notch pathway is inactive. Elucidating the mechanisms involved in the selection of Ascl1 targets will shed light on the pathways that control the switch from proliferation to differentiation during neural development (Castro, 2011).